Clarity chemiluminescent substrate

Protein samples were extracted using radioimmunoprecipitation assay (RIPA) buffer containing 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl and freshly added Protease and Phosphatase Inhibitors (Invitrogen). Protein concentrations were determined using Pierce™ BCA Protein Assay Kit (Thermofisher). 30 μg of protein from each sample was used in NuPAGE gel electrophoresis and XCell Western blot (Invitrogen). Proteins were revealed using the primary and secondary antibodies according to the manufacturer protocol (Supplementary Table 4), and bands were revealed using the Clarity Chemiluminescent Substrate (Biorad) and captured with X-ray films. ImageJ software was used to perform densitometry analyses. For IGF-1 dependent phosphorylation analyses, the monolayer of AMD and normal RPE were cultured in serum free media and starved in HBSS for 2hrs followed by incubation with IGF-1 (75 ng/ml) for the indicated times.

Protein samples were extracted in radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 0.5% sodium deoxycholate, and 1% SDS in 1x PBS), containing freshly added protease and phosphatase inhibitor cocktail tablets (Roche Applied Science, Indianapolis, IN, USA), 1x Protease Inhibitor Cocktail Set I (EMD Millipore), 1 mM sodium vanadate, 50 mM sodium fluoride and 1 mM PMSF (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were measured by Bradford assay (Bio-Rad, Hercules, CA, USA). Protein samples were analyzed using the NuPAGE electrophoresis and XCell western blot system (Invitrogen). Primary and secondary antibodies were used based on the manufacturer's instructions. Immunoreactive protein bands were visualized by the Clarity chemiluminescent substrate (Bio-Rad) followed by revealing with X-ray films. Densitometry was performed using the ImageJ software (https://ImageJ.nih.gov).

Reovirus treated and untreated cultured cells were collected, washed in PBS, and lysed in 150 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA and 1% Nonidet P-40 supplemented with a mixture of protease inhibitors (Roche, Basel, Switzerland). 400 micrograms total protein was pre-cleared and used to immune precipitate TLR3 protein using protein Agarose beads and TLR3 antibody (Antibody online # ABIN201783) for 24 hours at 4°C. The Agarose beads were washed thrice, eluted by boiling with Lameli buffer (Biorad 4x Laemmli Buffer #1610747) and run on a 12% SDS-PAGE. After blotting, membranes were probed with anti-TLR3 primary antibody (Antibody online # ABIN201783). Secondary antibodies were HRP-labeled, and detection was performed using the Clarity Chemiluminescent substrate (Bio-Rad # 1705061). Western blots were performed using standard procedures. Membranes were blocked with 5% milk in TBS containing 0.1% Tween 20, and incubated with antibodies specific for TLR3. Immunoreactive bands were visualized by chemiluminescence (# 1705061 Bio-Rad) clarity ECL western blotting detection kit.

The cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet-P40, 0.5% SDS, 10% glycerol, 1 mM dithiothreitol [DTT], 1 mM NaF, and ethylenediaminetetraacetic acid-free mini protease inhibitor cocktail [catalog no. 11873580001, Roche], pH 8.0) and scraped off the dish. The lysates were briefly incubated on ice, passed three times through a 25-gauge needle, incubated on ice for 15 min, and centrifuged at 20,000 × g for 30 min at 4 °C. For Western blot, the supernatant samples were mixed with sample buffer and boiled for 5 min. The proteins were then resolved by SDS-PAGE and transferred to nitrocellulose membranes (catalog no. 66485, Pall Corporation) using Trans Blot Turbo (catalog no. 1704270, Bio-Rad). Membranes were blocked with 5% milk in TBST (20 mM Tris-HCl, 150 mM NaCl [pH 7.6], and 0.1% Tween20) and probed with anti-AChR-α1 antibody (catalog no. 10613-1-AP, Proteintech), anti-rapsyn antibody (catalog no. ab156002, Abcam) or anti-tubulin antibody (catalog no. ab18251, Abcam) in 5% milk in TBST. After washing with TBST buffer, the membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase. For protein detection, we used Clarity chemiluminescent substrate (catalog no. 1705060, Bio-Rad).

Proteins were separated using 4–12% Bis Tris Plus 15 well Nupage BOLT gels in Novex Bolt Mini Gel Tanks (Life Technologies, CA, USA) and transfer was done in a Xcell II Bolt Module (Novex, CA, USA). The membrane was blocked using 2% casein in TBS and washed with TBST. The primary antibody; Mdr (G-1) mouse monoclonal IgG2b 200 and mouse anti-β actin were used as the antibody for β-actin, the reference protein. The antibody; HRP linked goat anti mouse IgG was used as the secondary antibody. The washed membrane was incubated in a BioRad clarity chemiluminescent substrate and enhancer was read and semi quantified using the BIORAD Chemidoc MPT imager with Image LabTM software.

Proteins were separated on 10% or 12% SDS-polyacrylamide gels, transferred to nitrocellulose membranes (Ambion), and western blot analyses were performed with specific primary antibodies and peroxidase-coupled secondary antibodies (Sigma). We used the Clarity chemiluminescent substrate (Biorad) and Biorad Chemidoc XRS camera to visualize the blot. Quantification was done using Image lab software (Biorad). We used anti-Mtq2-Trm112 and eRF1 antibodies raised in rabbit against purified recombinant yeast Mtq2-Trm112 complex and recombinant eRF1, respectively. Mouse monoclonal anti-PGK1 was purchased from Invitrogen. Antibodies against methylated eRF1 were raised in rabbit against the peptide HGRGGQSALRFA present in yeast eRF1, where the glutamine residue is N5-monomethylated (Eurogentec). These antibodies were then purified based on their retention on a column grafted with the methylated peptide but not on a column grafted with the unmethylated peptide.